User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/18
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June 18, 2012: Initial entry for PHIX174 research project
SC 17:54, 18 June 2012 (EDT)
The current log for this research project is linked to here: PHIX174_Research_Log_Jun-18-2012.txt. It will be updated at the beginning of each week. I am currently working on Characterization B and Hypothesis 2.
Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.
Previous attempts at cloning a UTR1-deGFP linker into pBEST-pA-BamHI//XhoI-T500 backbone have failed. Sequencing showed a systematic error, whereby a truncated ~100bp piece was ligated, instead of the full UTR1-deGFP linker. Re-digesting and purifying the backbone showed the same result. Therefore, I am focusing on the linker. My hypothesis is that the PCR was mis-primed, so I designed a different set of primers to make the BamHI-UTR1-deGFP-XhoI linker. They were received today.
Hypothesis 2: Gene L is necessary for phage propogation.
Over the weekend, I attempted whole plasmid PCR to amplify PHIX174 by whole plasmid PCR (non-mutagenic primers) by the method described in Chen and Ruffner. No amplification observed. One line at 45b observed, corresponding to self-hybridization of the primers.